Iron (Fe) is an abundant protein cofactor required for the activity of a myriad of proteins and consequently is essential for numerous cellular functions ranging from DNA synthesis to respiration. Therefore, the cell must ensure that a sufficient supply of Fe is available to these Fe-dependent proteins but, at the same time, excess Fe in the cell, which can lead to cytotoxic reactions, must be avoided. Although our understanding of Fe homeostasis has benefited from over half a century of study in numerous organisms, the molecular details concerning intracellular Fe trafficking are lacking. The Merchant group has developed Chlamydomonas reinhardtii as a reference organism for studying Fe metabolism in the context of poor Fe nutrition. As it is estimated that roughly one-third of the world's population suffers from symptomatic Fe deficiency and multiple human diseases are caused by mis-regulation of Fe homeostasis, understanding these mechanisms is crucial. One under-characterized response is the recycling of Fe from dispensable proteins when extracellular Fe is unavailable. How this process is regulated at the molecular level is not known. The goal of this project is to identify the trafficking pathways of Fe within the cell during Fe-limitation and discover proteins responsible for accomplishing and regulating Fe recycling. The specific aims of this project are three-fold. First, cells will be biochemically fractionated and distribution of Fe between Fe-utilizing and Fe-storage compartments in Fe-replete vs. -deficient conditions will be determined, especially in the context of carbon source utilization (respiration vs. photosynthesis). Second, reverse genetics will be employed to characterize the involvement of Fe-regulated genes predicted to be involved in Fe recycling. Characterization will be aided by determining the subcellular location of these proteins and how these genes are regulated by Fe. Third, a classical genetic screen will be implemented to discover novel components of Fe homeostasis that may not be regulated at the gene or mRNA level and, therefore, have escaped the notice of transcriptome studies. These aims were chosen to set a precedent for understanding the mechanism of regulated Fe trafficking in the cell, characterize genes known to be induced by Fe-limitation and discover novel genes involved in acclimating to Fe-limitation. In addition, this project has been designed to provide training with a wide-range of techniques and to gain expertise in working with a reference eukaryotic organism.